Multiplex recording of cellular events over time on CRISPR biological tape

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Science  15 Dec 2017:
Vol. 358, Issue 6369, pp. 1457-1461
DOI: 10.1126/science.aao0958
  • Fig. 1 Temporal recording in arrays by CRISPR expansion (TRACE).

    (A) Akin to an audio tape, temporal biological signals can be stored in DNA arrays within a cell population. (B) TRACE functions by first transforming an input biological signal to an altered abundance of trigger DNA (orange). This trigger DNA, alongside reference DNA (blue), is then recorded as spacers in genomic CRISPR arrays of a cell population in a unidirectional fashion, enabling capture of temporal information. (C) The pTrig trigger plasmid includes a mini-F origin for stable maintenance and an IPTG-inducible phage P1 replication system for copy number increase. PLac, Lac promoter. (D) qPCR measurement of pTrig relative copy number (log10 scale) in cells exposed to no IPTG or 1 mM IPTG for 6 hours. (E) The pRec recording plasmid includes an aTc-inducible E. coli Cas1 and Cas2 expression cassette. (F) Experimental induction scheme and CRISPR array sequencing approach. (o/n, overnight). (G) Cells with pRec or with pRec and pTrig were exposed to 100 ng/μL aTc and no or 1 mM IPTG and subjected to sequencing; resulting arrays with a single new spacer and identified source (genome, pRec, or pTrig) are plotted as a percentage of all measured CRISPR arrays. Error bars represent standard deviation of three biological replicates.

  • Fig. 2 Temporal recording of 4-day input profiles.

    (A) Cell populations were subjected to daily exposures over 4 sequential days (d1 to d4), constituting all 16 possible temporal signal profiles. (B) Resulting CRISPR arrays were sequenced with (black) and without (gray) a size-enrichment method. The frequencies (log10 scale) of unexpanded (un) and expanded arrays of different lengths (L1 to maximum detectable L5) are plotted. (C) Input profiles are grouped by number of pTrig inductions, and the percentage of pTrig spacers in each profile is displayed; red lines indicate means and standard deviations. (D) On the left, 50 L4 arrays sampled from the full data set for the input profile [on, on, off, off] are shown (shaded, pTrig spacer; unshaded, reference spacer; positions p1 to p4, 5′-to-3′ of array). Spacer incorporation can be analyzed across arrays of different lengths (L) and positions (p) as a heatmap displaying percentages of pTrig spacers detected at each location (right). (E) CRISPR arrays derived from recordings of all 16 temporal signal profiles. (F) The input signal profile (left) and corresponding L4 arrays (right, shown in reverse order to aid visual comparison) are displayed.

  • Fig. 3 Reconstructing temporal signal profiles and population lineages.

    (A) CRISPR array populations can be described as a frequency distribution consisting of all permutations of reference (R, blue) and trigger (T, orange) spacers for a given array length (L); L3 arrays are depicted. (B) As an example, for two distinct profiles with an equal number of inductions, observed (black) and model-predicted (white) L3 array-type frequencies are plotted; L3 positional averages are shown for reference (inset). (C) Euclidean distances between observed (rows) and model-predicted (columns) array-type distributions were calculated and normalized by row (L2, L3, and L4 array-type distributions are concatenated). The correct temporal signal profiles are indicated by white asterisks, and the models with minimum distance to the observed data are indicated by black outlines. (D) Number of profiles correctly classified using arrays L1 to L4 individually or arrays L2 to L4 together as in (C); the gray dashed line indicates the expected random classification (one of 16 correct). (E) A defined branching history was used in the temporal recording experiment. (F) The mapping locations for genomic spacers within L1 arrays were used as the sequence identity of the spacer. The Jaccard distances between all samples (1 minus the proportion of spacers shared between two samples) are displayed. Lineage reconstruction was performed using the Fitch-Margoliash method on this distance matrix and is displayed on the left; only one lineage is not fully differentiated (cells receiving induction on d1).

  • Fig. 4 Multiplex temporal recording with a barcoded sensor population.

    (A) The direct repeat (DR) of a CRISPR array can be barcoded to associate sensors with specific arrays; generated distal DR sequences with barcodes (bc) are shown. Sensors of copper, trehalose, and fucose were linked to the pTrig system and introduced into barcoded strains. The copper sensor uses a native promoter with endogenous transcription factor expression, whereas the trehalose and fucose sensors use an engineered transcription factor. (B) The three barcoded sensor strains were mixed and exposed to eight combinatorial inputs of the three chemicals; the resulting percentage of pTrig spacers for each barcoded sensor strain is displayed (average of three biological replicates). (C) The strain mixture was exposed to combinatorial inputs over 3 days. As an example, profile 5 is displayed, along with CRISPR arrays for each sensor (plotted as in Fig. 2, but the color map is rescaled for each sensor to aid visualization) and the resulting classification (correct, blue checkmark; incorrect, red X). (D) Of 512 (83) possible profiles, 16 were tested (six defined and 10 randomly generated); the resulting classification is shown as in (C) (black arrows indicate the time course, d1 to d3). (E) Single-channel classification accuracy. Profiles were classified for each sensor on the basis of L2 and L3 arrays; the gray dashed line indicates the expected random classification (two of 16 correct). (F) Multichannel classification accuracy. Predictions were considered across all three sensors, and the number classified correctly within a Hamming distance threshold is shown (black line) compared with the expected random classification (gray dashed line).

Supplementary Materials

  • Multiplex recording of cellular events over time on CRISPR biological tape

    Ravi U. Sheth, Sung Sun Yim, Felix L. Wu, Harris H. Wang

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
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    • Tables S1 to S5
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